The present invention relates to analyzing chemical assays and, more particularly, but not exclusively to cumulative differential photometric chemical assay identification.
Some chemical assays can be identified by monitoring their varying photometric properties in an ongoing chemical reaction (say in an on going Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), on a DNA (Deoxyribonucleic acid) Melting Reaction, or in another chemical reaction), as known in the art.
More specifically, crucial decisions in pre-implantation genetic diagnosis, infectious diseases, bioterrorism, forensics, and cancer research have increasingly depended on identification of specific DNA sequences, even down to alleles of single-copy genes in single cells.
The identification typically involves introduction of fluorescently active agents that emit or quench fluorescent light when connected in a weak bond, say to a specific DNA sequence, when disconnected from the weak bond, etc.
Melting Curve Analysis is often used, to map the hybridization temperature of two complementary DNA strands, or of a single DNA strand to a fluorescence emitting (or fluorescence quenching) sequence specific hybridizing probes (i.e. sequence specific fluorescently active agents).
The temperature may depend on the energy required to break base-base hydrogen bonding between two strands of DNA.
The energy is dependent on the strand's length, GC (Guanine-Cytosine) content, complementarity, etc.
PCR methods that monitor DNA melting with sequence specific fluorescently active agents have become popular in conjunction with real-time PCR. Because PCR produces enough DNA for fluorescent melting analysis, both amplification and analysis can be performed in a same reaction tube, thus providing a homogeneous, closed-tube system that requires no processing or separation steps.
In implementation, the tube is heated and photometers are used to measure fluorescent light in the reaction tube as a function of temperature. The fluorescent light in the reaction tube may also be measured post heating, as the temperature in the reaction tube gradually declines.
Conventional real-time PCR may permit rapid and quantitative identification of unique DNA targets (i.e. specific DNA sequences) on a double stranded DNA, but reactions typically slow down and plateau stochastically because re-annealing of the DNA's strands gradually outcompetes primer and probe binding to the strands, as know in the art.
Asymmetric PCR preferentially amplifies one strand of DNA. Asymmetric PCR potentially circumvents the problem of strand re-annealing, by using unequal primer concentrations. Depletion of the limiting primer during the exponential amplification of the PCR reaction results in linear synthesis of strands extended from the excess primer.
Although asymmetric PCR generates brighter signals than symmetric PCR does, asymmetric PCR is seldom used because it is much less efficient than conventional PCR, as described in further detail hereinbelow. Asymmetric PCR also requires extensive optimization to identify the proper primer ratios, the amounts of starting material, and the number of amplification cycles that can generate reasonable amounts of product for specific DNA sequences.
LATE (Linear after the exponential) PCR is a recently introduced technique.
LATE PCR was described by J. Aquiles Sanchez, Kenneth E. Pierce, John E. Rice, and Lawrence J. Wangh of the Biology Department of the Brandeis University, in an article published in PNAS (Proceedings of the National Academy of Sciences of the US), on Feb. 17, 2004, in Vol. 101, No. 7, on pages 1933-1938, entitled: “Linear-After-The-Exponential (LATE) PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis”.
While conventional symmetric PCR typically uses equimolar concentrations of two primers with similar melting points, conventional asymmetric PCR assays are inefficient and unpredictable, because they are designed using symmetric primers, without taking into account the effect of the actual primer concentrations on primer melting points.
LATE-PCR provides a rational approach to generating single-stranded DNA products based on knowledge of the primer-target hybridization equilibriums that drive asymmetric reactions. As a result, LATE-PCR may exhibit similar efficiency to symmetric PCR and enable the use of primers over a wide range of concentration ratios.
Under LATE-PCR conditions, the initial exponential phase of the reaction generates double-stranded amplicons until the limiting primer concentration falls abruptly and the reaction switches to synthesis of only excess primer strands.
In the case of real-time LATE-PCR, the amount of limiting primer is deliberately chosen such that the exponential phase of the reaction switches to the linear phase shortly after the reaction reaches detectability, i.e., at the CT value.
LATE-PCR therefore maintains the quantitative nature of real-time symmetric PCR assays, which is based on the CT values of the exponential phase of the reaction. Upon switching, the number of excess primer strands accumulated per cycle is proportional to the number of limiting primer strands present at the time of the switch.
LATE-PCR makes it possible to introduce a detection step distinct from the annealing step. The temperature of the detection, therefore, can be lowered to permit the use of low melting temperature probes with a greater allele-discrimination capacity and an improved signal-to-noise ratio.
Because the melting temperature of a low-melting temperature probe is well below the extension temperature of the reaction, saturating concentrations can be used to detect all of the single-stranded molecules produced.
The classification of chemical assays using any one of the above described methods includes a final step, in which an expert in the field manually examines a graph which represents fluorescence light measured through the chemical reaction, as a function of a physical parameter such as temperature.
The expert may further compare the examined graph with reference graphs, say for identifying occurrence of certain DNA sequences (say certain mutations), as known in the art.